Juvenile Hormone Hemolymph Titers Research Articles

Juvenile hormone mediates lipid storage in the oocytes of Dipetalogaster maxima

Triatomines are vectors of Chagas disease and important model organisms in insect physiology. “Kissing bugs” are obligatory hematophagous insects. A blood meal is required to successfully complete oogenesis, a process primarily controlled by juvenile hormone (JH). We used Dipetalogaster maxima as an experimental model to further understand the roles of JH in the regulation of vitellogenesis and oogenesis. A particular focus was set on the role of JH controlling lipid and protein recruitment by the oocytes. The hemolymph titer of JH III skipped bisepoxide increased after a blood meal. Following a blood meal there were increased levels of mRNAs in the fat body for the yolk protein precursors, vitellogenin (Vg) and lipophorin (Lp), as well as of their protein products in the hemolymph; mRNAs of the Vg and Lp receptors (VgR and LpR) were concomitantly up-regulated in the ovaries. Topical administration of JH induced the expression of Lp/LpR and Vg/VgR genes, and prompted the uptake of Lp and Vg in pre-vitellogenic females. Knockdown of the expression of LpR by RNA interference in fed females did not impair the Lp-mediated lipid transfer to oocytes, suggesting that the bulk of lipid acquisition by oocytes occurred by other pathways rather than by the endocytic Lp/LpR pathway. In conclusion, our results strongly suggest that JH signaling is critical for lipid storage in oocytes, by regulating Vg and Lp gene expression in the fat body as well as by modulating the expression of LpR and VgR genes in ovaries.

JH biosynthesis and hemolymph titers in adult male Aedes aegypti mosquitoes

Juvenile hormone (JH) is a major hormonal regulator in insects. In Aedes aegypti females, JH signals the completion of the ecdysis to the adult stage and initiates reproductive processes. Although the regulation of JH synthesis and titer in Ae. aegypti females has been extensively studied, relatively little is known about changes of JH synthesis and titers in male mosquitoes, as well as on the roles of JH controlling male reproductive biology. A better understanding of male mosquito reproductive biology, including an improved knowledge of the hormonal control of reproduction, could increase the likelihood of success of male-targeting vector control programs. Using a high performance liquid chromatography coupled to electrospray tandem mass spectrometry method, we measured JH biosynthesis and hemolymph levels in male mosquitoes during pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers. Synthesis of JH III was very low in late pupae, significantly increased during the first 24 h after adult eclosion, and then remained relatively constant during the first six days after adult eclosion. Feeding high sugar diets resulted in an increase of JH synthesis and titers, and starvation significantly decreased JH synthesis, but this effect could be reversed by changing the males back to a high sugar diet. JH synthesis rates were similar in virgin and mated males, but hemolymph JH levels were different in well-nourished virgin and mated males. Starvation resulted in a significant reduction in insemination rates; with well-nourished males inseminating 2 times more females than water-fed. Giving a 20% sugar meal for 24 h to those mosquitoes that were previously starved for 6 days, caused a significant rise in insemination rates, restoring them to levels similar to those recorded for 20% fed males. These results suggest that nutrition plays a role on male fecundity, and this effect might be mediated by JH.

Juvenile Hormone Regulates Extreme Mandible Growth in Male Stag Beetles

The morphological diversity of insects is one of the most striking phenomena in biology. Evolutionary modifications to the relative sizes of body parts, including the evolution of traits with exaggerated proportions, are responsible for a vast range of body forms. Remarkable examples of an insect trait with exaggerated proportions are the mandibular weapons of stag beetles. Male stag beetles possess extremely enlarged mandibles which they use in combat with rival males over females. As with other sexually selected traits, stag beetle mandibles vary widely in size among males, and this variable growth results from differential larval nutrition. However, the mechanisms responsible for coupling nutrition with growth of stag beetle mandibles (or indeed any insect structure) remain largely unknown. Here, we demonstrate that during the development of male stag beetles (Cyclommatus metallifer), juvenile hormone (JH) titers are correlated with the extreme growth of an exaggerated weapon of sexual selection. We then investigate the putative role of JH in the development of the nutritionally-dependent, phenotypically plastic mandibles, by increasing hemolymph titers of JH with application of the JH analog fenoxycarb during larval and prepupal developmental periods. Increased JH signaling during the early prepupal period increased the proportional size of body parts, and this was especially pronounced in male mandibles, enhancing the exaggerated size of this trait. The direction of this response is consistent with the measured JH titers during this same period. Combined, our results support a role for JH in the nutrition-dependent regulation of extreme mandible growth in this species. In addition, they illuminate mechanisms underlying the evolution of trait proportion, the most salient feature of the evolutionary diversification of the insects.

Hormones in the Field: Evolutionary Endocrinology of Juvenile Hormone and Ecdysteroids in Field Populations of the Wing‐Dimorphic CricketGryllus firmus

Virtually no published information exists on insect endocrine traits in natural populations, which limits our understanding of endocrine microevolution. We characterized the hemolymph titers of juvenile hormone (JH) and ecdysteroids (ECDs), two key insect hormones, in field-collected short-winged, flightless (SW) and long-winged, flight-capable (LW(f)) morphs of the cricket Gryllus firmus. The JH titer exhibited a dramatic circadian rhythm in the LW(f) morph but was temporally constant in the flightless SW morph. This pattern was consistent in each of three years; in young, middle-aged, and older G. firmus; and in three other cricket species. The ECD titer was considerably higher in SW than in LW(f) females but did not exhibit temporal variation in any morph and did not differ between male morphs. JH and ECD may control different aspects of the morph-specific trade-off between nocturnal dispersal and reproduction. Results confirm and extend laboratory studies on young female G. firmus; most, but not all, important aspects of morph-specific differences in JH and ECD titers can be extrapolated from field to laboratory environments and vice versa. Hormone titers in Gryllus are more complex than those proposed in evolutionary endocrine models. Directly measuring hormone titer variation remains a fundamentally important task of insect evolutionary endocrinology.

Early events in adult eye development of the moth, Manduca sexta

The eye imaginal disc of Manduca sexta is created early in the final larval instar from the adult eye primordium, which is composed of fully differentiated cells of the larval head capsule epidermis. Concomitant with the down-regulation of the larval epidermal program, expression of broad, a marker of pupal commitment, is activated in the primordium. The cells then detach from the cuticle, fold inward, and begin to proliferate at high levels to produce the inverted, eye imaginal disc. These and other events that begin on the first day of the final larval instar appear to mark the initiation of metamorphosis. Little is known about the endocrine control of the initiation of metamorphosis in any insect. The hemolymph titer of juvenile hormone (JH) declines to low levels during this period and the presence of JH is sufficient to repress development in cultured eye primordia. However, maintenance of JH at high levels in vivo by treatment with long-lasting JH mimics has no apparent effect on early steps in eye imaginal disc development. We discuss our findings in the context of the endocrine control of metamorphosis. The initiation of metamorphosis in Manduca, and perhaps a wide range of insect species, appears to involve the overcoming of JH repression by an unidentified, nutrient-dependent, hormonal factor.

The role of nutrition in creation of the eye imaginal disc and initiation of metamorphosis in Manduca sexta

With the exception of the wing imaginal discs, the imaginal discs of Manduca sexta are not formed until early in the final larval instar. An early step in the development of these late-forming imaginal discs from the imaginal primordia appears to be an irreversible commitment to form pupal cuticle at the next molt. Similar to pupal commitment in other tissues at later stages, activation of broad expression is correlated with pupal commitment in the adult eye primordia. Feeding is required during the final larval instar for activation of broad expression in the eye primordia, and dietary sugar is the specific nutritional cue required. Dietary protein is also necessary during this time to initiate the proliferative program and growth of the eye imaginal disc. Although the hemolymph titer of juvenile hormone normally decreases to low levels early in the final larval instar, eye disc development begins even if the juvenile hormone titer is artificially maintained at high levels. Instead, creation of the late-forming imaginal discs in Manduca appears to be controlled by unidentified endocrine factors whose activation is regulated by the nutritional state of the animal.

Endocrine changes in maturing primary queens of Zootermopsis angusticollis

Termite queens are highly specialized for reproduction, but little is known about the endocrine mechanisms regulating this ability. We studied changes in the endocrinology and ovarian maturation in primary reproductive females of the dampwood termite Zootermopsis angusticollis following their release from inhibitory stimuli produced by mature queens. Winged alates were removed from their natal nest, manually dewinged, then paired in an isolated nest with a reproductive male. Development was tracked by monitoring ovarian development, in vitro rates of juvenile hormone (JH) production by corpora allata, and hemolymph titers of JH and ecdysteroids. The production rate and titer of JH were positively correlated with each other but negatively correlated with ecdysteroid titer. Four days after disinhibition, JH release and titer decreased while ecdysteroid titer increased. The new levels persisted until day 30, after which JH increased and ecdysteroids decreased. Fully mature queens had the highest rates of JH production, the lowest ecdysteroid titers, and the greatest number of functional ovarioles. The results support the hypothesis that JH plays a dual role in termite queens depending on their stage of development; an elevated JH titer in immature alates may maintain reproductive inhibition, but an elevated JH titer in mature queens may stimulate ovarian activity. The decline in JH production and the elevation in ecdysteroid titer correspond to a period of physiological reorganization and activation. The specific function of ecdysteroids is unknown but they may help to modulate the activity of the corpora allata.

Social stimuli affect juvenile hormone during breeding in biparental burying beetles (Silphidae: Nicrophorus)

Extended biparental care is rare in insects but provides an excellent opportunity to investigate the interaction between the endocrine system and the physical and social environment in the regulation of this behavior. Burying beetles ( Nicrophorus spp.) have facultative biparental care and depend on locating a small vertebrate carcass that they bury and prepare as food for their young. Commonly, both male and female Nicrophorus orbicollis remain in the burial chamber after eggs hatch to feed and guard the larvae. In both sexes, juvenile hormone (JH) rises rapidly in response to the discovery and assessment of the carcass; it returns to near baseline in 24 h; then in females it reaches very high titers at the onset of maternal care. In this paper, we investigate some social (presence of a mate, mating history, larval age) and environmental (carcass size) factors that may affect this endocrine profile. For females, neither the presence of a mate nor mating status (i.e., virginity) affected the initial rise of JH. However, the absence of a mate significantly depressed the JH rise in males. Eighty-seven percent of the single males buried the carcass like paired males but 87% also released pheromones to attract a mate. JH hemolymph titers in females whose broods were replaced every 24 h with newly hatch larvae were significantly higher than those of females rearing aging broods. Lastly, even though larger carcasses took longer to bury and prepare and oviposition was delayed, neither JH titers nor speed of ovarian development was affected by carcass size.

Evidence for Reproductive Diapause in the Fritillary <I>Speyeria idalia</I> (Lepidoptera: Nymphalidae)

Reproductive characteristics of a northeastern Kansas population of the fritillary Speyeria idalia (Drury) were studied. Hemolymph juvenile hormone (JH) titers, ovarian development, and fat body utilization were monitored weekly in adult females over their entire 1997 flight period, which extended from mid-June to early October. Dissections of female reproductive systems revealed that S. idalia females mate just once, soon after they emerge in mid- to late June. Gas chromatographic-mass spectrometric determinations of juvenile hormone suggested that they undergo a reproductive diapause through mid-August related to the absence of or very low titers of juvenile hormone. Oogenesis and fat body depletion do not commence until late August/early September, soon after which oviposition occurs. The onset of oogenesis coincides with a rapid rise in hemolymph titers of JH I, JH II, and JH III. The predominant juvenile hormone homolog was JH II, but both JH I and JH III exhibited smaller, concomitant peaks. Four fundamentally different lepidopteran reproductive strategies have been recognized based on various reproductive characteristics and the type of gonadotropic hormones used to stimulate oogenesis. Speyeria idalia exhibits a type of reproductive strategy that has not been documented in Lepidoptera, typified by protandry, female monandry, long-lived (>8 wk) females that feed throughout their adult lives, greatly delayed oogenesis that occurs late in adult life, and apparent juvenile hormone control of gonadotropic processes. This reproductive strategy appears to be an adaptation to the phenology of larval host plants, namely coordinating the life cycle with that of the seasonally restricted violets on which larval survival of this monophagous species depends.

Genetic and diurnal variation in the juvenile hormone titer in a wing-polymorphic cricket: implications for the evolution of life histories and dispersal.

The wing-polymorphic cricket, Gryllus firmus, contains (1) a flight-capable morph (LW(f)) with long wings and functional flight muscles, (2) a flightless morph with reduced wings and underdeveloped flight muscles (SW), and (3) a flightless morph with histolyzed flight muscles but with fully developed wings (LW(h)). The LW(f) morph differed genetically from the SW morph and phenotypically from the LW(h) morph in the size of flight muscles, ovarian growth during the first week of adulthood, and the hemolymph titer of juvenile hormone (JH). This is the first study to document that phenotypes that differ genetically in morphological aspects of dispersal capability and in ovarian growth also differ genetically in the titer of a hormone that potentially regulates those traits. The JH titer rose 9-100-fold during the photophase in the flight-capable LW(f) morph but did not change significantly during this time in either flightless morph. Prolonged elevation of the in vivo JH titer in flight-capable females, by topical application of a hormone analogue, caused a substantial increase in ovarian growth and histolysis of flight muscles. The short-term, diurnal rise in the JH titer in the dispersing morph may be a mechanism that allows JH to positively regulate nocturnal flight behavior, while not causing maladaptive histolysis of flight muscles and ovarian growth. This is the first demonstration of naturally occurring, genetically based variation for diurnal change in a hormone titer in any organism.

The effect of queen-worker conflict on caste determination in the bumblebee Bombus terrestris

Endocrine analyses were used to investigate the well-known association between queen production and the onset of worker reproduction (termed the competition phase, CPh) in Bombus terrestris. Larvae that reached the age of 5 days before the CPh had a worker-like profile: low juvenile hormone (JH) biosynthesis rates and low JH hemolymph titers. In contrast, larvae that reached the age of 5 days during the CPh had a queen-like profile: high JH biosynthesis rates and high hemolymph JH levels. Larval fate could be manipulated by transplanting egg cells into host colonies with different social structures. There was a steep rise in JH production in larvae transplanted into colonies near or during the CPh. This indicates that during colony development, larvae switch from the ”worker developmental pathway” to the ”queen developmental pathway,” and that the switch is socially regulated. In small rearing groups, larvae reared with queens before the CPh developed into workers, whereas those reared with queens after the CPh developed into queens. Variation in worker type (naive or experienced) did not affect caste determination. Therefore, we hypothesize that queens produce a pheromone that directly inhibits queen differentiation by larvae. We also present two alternative scenarios that explain the timing of gyne production in B. terrestris, one based on ecological constraints and the other based on queen-worker competition.

Lipophorin of female Blattella germanica (L.): characterization and relation to hemolymph titers of juvenile hormone and hydrocarbons

High density lipophorin (HDLp) from the hemolymph of the German cockroach, Blattella germanica (L.) (Family Blattellidae), has an apparent molecular weight of 670 kDa, with an isoelectric point of 7.0 and a density of 1.109 g/ml. It is composed of two subunits, apolipoprotein-I (212 kDa) and apolipoprotein-II (80 kDa), and consists of 51.4% lipid, 46.2% protein and 2.4% carbohydrate. Hydrocarbons constitute 42.2% of the total lipids which also contain diacylglycerol, cholesterol and phospholipid. Lipophorin is rich in the amino acids glutamic acid, aspartic acid, lysine, valine, and leucine. Specificity of a polyclonal antibody was demonstrated by Western blotting and Ouchterlony immunodiffusion: the antiserum recognized native HDLp and apolipoprotein-I, but not apolipoprotein-II, purified vitellin, or other hemolymph proteins. It also recognized a protein in the hemolymph of Supella longipalpa (Blattellidae) but did not cross-react with hemolymph proteins from Periplaneta americana (Blattidae) or Diploptera punctata (Blaberidae). An enzyme-linked immunosorbent assay was developed to measure the HDLp titer in the hemolymph of adult females. The titer of HDLp, a juvenile hormone binding protein, exhibited no clear relationship to the changing titer of juvenile hormone in hemolymph. The hemolymph titer of hydrocarbon, which is also carried by HDLp, showed some functional relation to the concentration of HDLp in the hemolymph. Because it concurrently serves multiple functions in insect development and reproduction, lipophorin titer might covary with the titers of lipid ligands that occur at high concentrations and require extensive shuttling through the hemolymph.

Development changes in juvenile hormone and juvenile hormone acid titers in the hemolymph and in-vitro juvenile hormone synthesis by corpora allata of the silkworm, Bombyx mori

A simple method was developed to quantify hemolymph juvenile hormone (JH) and JH acid in hemolymph extracts from Bombyx mori with an established radioimmunoassay (RIA) for JH I. When various organic solvent extracts of hemolymph were assayed by RIA, levels of non-specific binding of the labeled ligand in the assay were determined to be greater than 50% of the maximum amount of the label bound by the antiserum. When hemolymph was diluted with methanol:water:8.4 N ammonium hydroxide (10:9:1) and extracted with isooctane, non-specific binding was only 50% higher than control levels obtained with the assay buffer alone. The organic phase contained only JH and aqueous phase, JH acid. Consequently, this extraction method was used to prepare samples for RIA and enabled the separate measurement of JH and JH acid in hemolymph. With this method, changes in the hemolymph titers of JH and JH acid were determined from the third instar through early pupal stage of Bombyx mori. Changes in the in vitro secretory activity of corpora allata and brain–corpora cardiaca–corpora allata complexes from fifth instar larvae were also determined by using JH I RIA of the incubation medium.

Juvenile hormone-mediated reproduction in burying beetles: From behavior to physiology

The burying beetle, Nicrophorus orbicollis, is the first beetle exhibiting parental care for which endocrinological studies have been initiated. Burying beetles bury and prepare small vertebrate carcasses as a breeding resource for their offspring. After emergence as an adult, hemolymph titers of juvenile hormone and ovarian size increase in concert for 2–3 weeks, and both plateau until an appropriate resource is discovered. Upon finding a suitable carcass, titers of juvenile hormone increase extremely rapidly (<20 min), and within 18 h ovarian mass increases threefold and oviposition begins. This rapid reproductive development is hypothesized to be selected by the intense competition for these protein-rich but quickly deteriorating resources. Burying beetle females exhibit an additional juvenile hormone surge at the time young hatch. This peak in juvenile hormone is hypothesized to be associated with either the considerable behavioral demands which accompany care of young larvae or with a female's willingness to oviposit a replacement clutch should brood failure occur early in the care-giving phase. Parental care has evolved in at least 16 additional families within the Coleoptera. Comparative studies of nonparental and parental groups will be important for understanding how the physiology of ancestral groups affects the evolution of complex social behavior. Arch. Insect Biochem. Physiol. 35:479–490, 1997. © 1997 Wiley-Liss, Inc.

Juvenile hormone activation of gene transcription in locust fat body

Transcription of a vitellogenin gene ( VgB) was measured in nuclei isolated from fat body of adult female locusts, to study its relations to juvenile hormone (JH). During the first ovarian cycle, there was a correlation between the rise and fall in the hemolymph titer of JH and the rate of Vg gene transcription. At the oviposition stage, when the JH titer fell sharply, Vg transcription fell almost to zero; both JH titer and Vg transcription then rose again in the subsequent cycle. Administration of the JH analog, pyriproxyfen, to ethoxyprecocene-pretreated locusts was followed, after a lag of about 12 h, by initiation and rapid rise in Vg transcription. Data on transcription of the 21K and apoLpIII genes, although imprecise because of short probe sequences, are consistent with the induction of 21K protein and lack of effect on apoLpIII protein by JH. Total transcription in fat body nuclei was also elevated after pyriproxyfen administration to precocene-treated locusts but declined during the vitellogenic cycle, suggesting that rRNA synthesis is regulated by both JH and other cellular factors.

Rapid elevation of juvenile hormone titer during behavioral assessment of the breeding resource by the burying beetle, Nicrophorus orbicollis

Female burying beetles ( Nicrophorus orbicollis) rapidly complete ovarian maturation upon discovering a suitable carrion resource for breeding. In this study, we examined changes in hemolymph titers of juvenile hormone (JH) over the first 30 days of adult female life, and in response to the discovery of a mouse carcass. Levels of JH were found to increase gradually over the first 20 days, and then increased abruptly within 24 h of discovery of a carcass. Changes in JH titer were correlated with increases in ovarian mass and length of terminal oocytes. To more precisely determine the timing of the endocrine response to a carcass, hemolymph titers of JH were measured 2, 10, 20 and 60 min after carcass discovery. Titers of JH were significantly elevated (112% over controls) in just 10 min. To confirm this resource discovery-related increase in JH, hemolymph samples were taken from the same individuals both 2 days before, and 10 min after, discovery of a carcass. Again, JH titers rose significantly (170%) in beetles 10 min after carcass discovery. Prominent behaviors observed during the 10-min period following discovery included palpating, lifting, walking around the carcass and making forays into the surrounding soil. Feeding did not occur. These results suggest that the rapid JH surge in female burying beetles is triggered by information obtained during behavioral assessment of a breeding resource, and not by mating or feeding cues. The association between ovarian development and JH titer further suggests a role for JH in co-ordinating reproduction in a temporally and spatially unpredictable environment.

Reproduction in worker honey bees is associated with low juvenile hormone titers and rates of biosynthesis

Three experiments were performed to determine the role of juvenile hormone (JH) in worker reproduction in queenless colonies of honey bees. In Experiment 1, egg-laying workers had low hemolymph titers of JH, as did bees engaged in brood care, while foragers had significantly higher titers. Experiment 2 confirmed these findings by demonstrating that laying workers have significantly lower rates of JH biosynthesis than foragers do. In Experiment 3, ovary development was inhibited slightly by application of the JH analog methoprene to 1-day-old bees, but was not affected by application to older bees, at least some already displaying egg-laying behavior. These results, which are consistent with earlier findings for queen honey bees, are contrary to a common model of insect reproduction, in which elevated JH titers trigger ovary development, which then leads to oviposition. Previous experiments have demonstrated that JH regulates nonreproductive behavior in workers that is associated with colony division of labor; perhaps this function is incompatible with a traditional role for JH in reproduction.

Caste and metamorphosis: Hemolymph titers of juvenile hormone and ecdysteroids in last instar honeybee larvae

Juvenile hormone (JH) and ecdysteroid titers are critical factors for caste development and metamorphosis in the last larval instar of the honeybee, Apis mellifera. Two highly sensitive radioimmunoassays were used for the determination of these hormones in the hemolymph. For juvenile hormone, which is of prime importance for the control of caste development in honeybees, our data show a caste-specific peak in queen larvae of the early fifth instar. A second peak appears in prepupae of both castes which probably is responsible for the regulation of the pupal moult. A single peak of ecdysteroids was observed in prepupae of both castes. In queens, however, the titer increases distinctly earlier than in worker larvae. The ecdysteroid composition of this prepupal peak was determined by high-performance liquid chromatography separation followed by radioimmunoassay. Makisterone A proved to be the main ecdysteroid compound, but 20-hydroxyecdysone was also found in significant amounts.

Brain-mediated 20-hydroxyecdysone regulation of juvenile hormone synthesis by the corpora allata of the tobacco hornworm, Manduca sexta

In the latter part of the last larval instar (Days 6–7) of Manduca sexta an increase in the hemolymph titer of juvenile hormone (JH) occurs which is involved in directing the pharate pupal development elicited by the large increase in the ecdysteroid titer on Days 7–8 of the instar. A time course analysis on Days 3–4 of the instar of both the first hemolymph ecdysteroid titer peak, which is responsible for wandering behavior and pupal commitment, and of the capacity of the corpora allata (CA) to synthesize JH I and III in vitro revealed a temporal correlation between the increasing ecdysteroid titer and a following increase in CA activity. The temporal arrangement of these increases suggested that ecdysteroids, i.e., 20-hydroxyecdysone, may elicit the increase in CA activity which contributes to the second increase in the JH titer during the latter part of the instar. Regulation of the CA by 20-hydroxyecdysone was investigated in vitro by assessing the effect of this ecdysteroid on JH I and III synthesis by CA incubated either as a complex with the brain-corpora cardiaca or as isolated glands. Dose-response analyses revealed that 20-hydroxyecdysone indeed had a stimulatory effect on JH synthesis by the CA, but only when the glands were complexed with the brain-corpora cardiaca. The effective concentration of 20-hydroxyecdysone was the same as the hemolymph concentration of the hormone at this time. Thus it appears that the increase in CA activity between Days 5 and 7 of the last larval instar is elicited in response to the commitment peak of 20-hydroxyecdysone, acting indirectly via the braincorpora cardiaca. These results constitute the first direct demonstration of interhormonal regulation between the ecdysteroids and juvenile hormones in an insect, and suggest that these hormones act to regulate postembryonic development not only at the level of the target tissue but by regulating the synthesis of the hormones by the endocrine glands themselves.

Reassessment of the critical periods for prothoracicotropic hormone and juvenile hormone secretion in the larval molt of the tobacco hornworm Manduca sexta

The ligation studies by which critical periods for prothoracicotropic hormone (PTTH), juvenile hormone (JH) and ecdysone were determined for the last larval molt in Manduca sexta are reviewed in relation to recent experimental observations on Manduca larvae. Our two findings which challenged the accepted interpretation of these critical period determinations were: (1) Larval molting in vitro of the crochet epidermis from fourth instar larvae required ecdysone but not JH. (2) The hemolymph titer of JH was high and relatively constant before and during the period of PTTH secretion in the fourth instar. We present further experimental results bearing on the endocrine requirements for larval molting. Uninjected fourth instar larvae neck-ligatured prior to the supposed PTTH critical period initiated metamorphosis precociously. Immediate injection of ecdysone into such neck-or abdomen-ligatured larvae resulted in a larval molt. When the ecdysone injection was delayed, the proportion showing a larval response declined. Juvenile hormone injections (JHI) at the time of neck ligation prevented this loss of response to injected ecdysone. Delayed injections of subthreshold amounts of ecdysone into neck-ligatured larvae caused a delay in the onset of metamorphosis relative to uninjected controls. Additional ligation studies throughout the initiation of larval molting suggested that the prothoracic glands were increasing secretory activity during this period. On the basis of this evidence we have reinterpreted the ligation data for Manduca larvae as follows: the supposed PTTH critical period corresponds to attainment of subthreshold secretory activity of the prothoracic glands; the supposed JH critical period identifies onset of full secretory activity and delimits the physiological PTTH critical period for larval molting.